Tumor cells are deficient in mechanisms which normally protect the cell against free radical damage. They have low levels of superoxide dismutase, glutathione peroxides and catalase. Tumor cells might be expected to show an increased sensitivity to drugs which increase in intracellular formation of free radicals. Free radicals are formed by several classes of antitumor drugs including quinones, quinoneimines, nitrocompounds and pyrrolizidine alkaloid N-oxides. However, a relationship between drug-induced free radical formation and cytotoxicity has yet to be demonstrated. Evidence for such a relationship would be the unambiguous detection of free radicals in a cell in an amount proportional to drug cytotoxicity. We will use electron spin resonance (esr) spectroscopy and a variety of biochemical techniques to measure anticancer drug-induced free radical formation by tumor cells and relate it to antitumor activity measured by the stem cell colony forming assay and to non-specific cytotoxicity measured by the log-growth inhibition assay. It is a widely accepted view that anticancer drugs exert a cytotoxic effect through an effect on the nucleus. This has not been conclusively established and some drugs, particularly those that form reactive intermediates, might exert an effect on other sites within the cell. We will use bacterial plasmids and reconstituted nuclear and cytoplasmic components of mammalian cells exposed to drug under different conditions to determine the site in the cell where the drugs exert their cytotoxic effect. Pulse radiolysis and esr spectroscopy will be used to study the properties of antitumor drug free radicals including radical lifetimes, one-electron reduction potentials, reactivity with oxygen and interaction of the free radicals with DNA and lipid membranes. We have found that blood from human subjects can reduce some antitumor quinone drugs to esr detectable free radicals. Blood from cancer patients shows a wider range and, as a group, an increased ability to form free radicals than blood from healthy individuals. We will study mechanism of free radical formation by blood and, in animals, study the possibility that drug-induced organ damage could release factors into the blood responsible for drug dependent radical formation. We will study free radical formation by several anticancer drugs with blood from patients receiving the drugs as part of their normal chemotherapy and relate radical formation to the toxic or therapeutic effects of the drug.